title: Miniature short hairpin RNA screens to characterize antiproliferative drugs.
abstract: The application of new proteomics and genomics technologies support a view in which few drugs act solely by inhibiting a single cellular target. Indeed, drug activity is modulated by complex, often incompletely understood cellular mechanisms. Therefore, efforts to decipher mode of action through genetic perturbation such as RNAi typically yields "hits" that fall into several categories. Of particular interest to the present study, we aimed to characterize secondary activities of drugs on cells. Inhibiting a known target can result in clinically relevant synthetic phenotypes. In one scenario, drug perturbation could, for example, improperly activate a protein that normally inhibits a particular kinase. In other cases, additional, lower affinity targets can be inhibited as in the example of inhibition of c-Kit observed in Bcr-Abl-positive cells treated with Gleevec. Drug transport and metabolism also play an important role in the way any chemicals act within the cells. Finally, RNAi per se can also affect cell fitness by more general off-target effects, e.g., via the modulation of apoptosis or DNA damage repair. Regardless of the root cause of these unwanted effects, understanding the scope of a drug's activity and polypharmacology is essential for better understanding its mechanism(s) of action, and such information can guide development of improved therapies. We describe a rapid, cost-effective approach to characterize primary and secondary effects of small-molecules by using small-scale libraries of virally integrated short hairpin RNAs. We demonstrate this principle using a "minipool" composed of shRNAs that target the genes encoding the reported protein targets of approved drugs. Among the 28 known reported drug-target pairs, we successfully identify 40% of the targets described in the literature and uncover several unanticipated drug-target interactions based on drug-induced synthetic lethality. We provide a detailed protocol for performing such screens and for analyzing the data. This cost-effective approach to mammalian knockdown screens, combined with the increasing maturation of RNAi technology will expand the accessibility of similar approaches in academic settings.
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
authors.forename: Saranya
authors.initials: S
authors.lastname: Kittanakom
authors.forename: Anthony
authors.initials: A
authors.lastname: Arnoldo
authors.forename: Kevin R
authors.initials: KR
authors.lastname: Brown
authors.forename: Iain
authors.initials: I
authors.lastname: Wallace
authors.forename: Tada
authors.initials: T
authors.lastname: Kunavisarut
authors.forename: Dax
authors.initials: D
authors.lastname: Torti
authors.forename: Lawrence E
authors.initials: LE
authors.lastname: Heisler
authors.forename: Anuradha
authors.initials: A
authors.lastname: Surendra
authors.forename: Jason
authors.initials: J
authors.lastname: Moffat
authors.forename: Guri
authors.initials: G
authors.lastname: Giaever
authors.forename: Corey
authors.initials: C
authors.lastname: Nislow
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D001549:Benzamides / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D016044:Fusion Proteins, bcr-abl / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D000068877:Imatinib Mesylate; D016086:Lentivirus / Q000235:genetics; D008904:Miniaturization; D010879:Piperazines / Q000494:pharmacology*; D011506:Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D019009:Proto-Oncogene Proteins c-kit / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D001549:Benzamides; D010879:Piperazines; D011506:Proteins; D011743:Pyrimidines; D034741:RNA, Small Interfering; D000068877:Imatinib Mesylate; D019009:Proto-Oncogene Proteins c-kit; D016044:Fusion Proteins, bcr-abl
keywords: next-generation sequencing; shRNA screening; synthetic lethality